Control of Gene Expression
Describe the process of catabolite repression of the lac operon by glucose
"Catabolite repression:" when glucose levels are low, ATP is converted into cyclic AMP (cAMP) CRP (cAMP receptor protein) is a transcription factor that activates RNA polymerase on the lac operon. It requires the binding of cAMP to be active. When glucose levels are high, cAMP is not created and activation of RNA polymerase doesn't happen.
Describe the lac operon in E.coli (what activates/inactivates it) Name the three enzymes on the lac operon, name their corresponding genes, and name their functions. All three of these genes are transcribed into a single strand of _____________ mRNA. What does this word mean?
*Substrate of lac operon: lactose. If this substrate is around, it binds to the operon's repressor to inactivate it and turn the lac genes ON. *Products of lac operon: glucose and galactose. If these products are around, the operon's repressor is active and lac genes are OFF. The gene for beta-galactosidase is lac Z; beta-galactosidase breaks lactose down into glucose and galactose. The gene for permease is lac Y; permease brings lactose into the cell across the membrane The gene for acetylase is lac a; acetylase's function is unknown. All 3 genes are transcribed into a strand of polycistronic mRNA. *Polycistronic mRNA is a type of mRNA that can encode more than one polypeptide separately within the same RNA molecule
Name some therapeutic developments that target gene expression
1. Deep sequencing or RNA-Seq to identify disease-specific gene expression abnormalities 2. Liquid biopsy on areas that can't be biopsied by conventional methods like the brain or lungs: take 5mL of blood and try to find what tumor biomarkers are present in the blood 3. Target specific mRNA to reduce expression of certain genes 4. CRISPR can correct disease-causing mutations in the genome
Describe the three DNA binding motifs that help transcription factors recognize enhancers
1. Helix-Turn-Helix motif: found in tryptophan repressor, phage I repressor, CRP, and the homeodomain of mammalian proteins. Binds DNA as a dimer. 2. Leucine Zipper motif: two alpha-helical DNA-binding domains dimerize through their alpha-helical leucine zipper region, forming an inverted Y-shaped structure. Each arm of the Y is formed from a single alpha-helix that mediates binding to a specific DNA sequence in the major groove of DNA. Leucine is hydrophobic. 3. Zinc-Finger motif: Cys-Cys-His-His with Zinc in the middle
Describe six steps at which gene expression can be controlled, and when in the process these steps occur.
1. Transcriptional: (while primary mRNA transcript is made in nucleus) 2. RNA processing control: (while mRNA is made in nucleus) 3. RNA transport control: (while mRNA is being moved to cytosol) 4. mRNA degradation control: (while certain mRNAs are inactivated) OR 5. Translational control (while protein is synthesized in cytosol) 6. Protein activity control (while certain proteins are inactivated)
What three functions do transcription factors have to affect gene expression?
Activate, inhibit, silent
What is an enhancer? What is a transcription factor? How does it recognize an enhancer?
An enhancer is a piece of DNA with a conserved sequence that alters gene expression by binding transcription factors. A transcription factor is a protein that can recognize the enhancer's conserved sequence through DNA binding motifs.
Describe what an operon is What induces/supresses transcriptional regulation of operons?
An operon is a transcriptional unit that organizes bacterial genes involved in the same metabolic pathways . Transcriptional regulation of operons can be induced by the substrate or repressed by the product of the encoded enzymes by the operon.
Beta-galactosidase is produced in what carbon source? Glucose and lactose Lactose without glucose Glucose without lactose
Beta-galactosidase is produced with lactose and without glucose.
Where does gene inactivation by DNA methylation tend to take place?
CG Islands CG sequences are unevenly distributed in the genome. They are clustered in islands, about 1000 to 2000 nucleoties long, near promoters of genes. Methylation causes stable transcriptional repression. The CG islands of active genes are kept in a dimethylated state. In inactive genes, the Cs are activated.
What does combinatorial expression of transcription factor proteins allow?
Combinatorial expression of transcription factor proteins allows differentiation of cells into different cell types.
Describe DNA methylation's effects on a gene
DNA methylation is the covalent addition of a methyl group on cytosine of CpG (cytosine-guanidine) dinucleotides. In eukaryotic organisms, DNA methylation is catalyzed by SAM dependent DNA cytosine-5-methyltransferase, which catalyzes the transfer of a methyl group from SAM to form methylcytosine. *Repression by inhibition by preventing TF binding
Name the family of steroid receptor transcription factors (six of them) and what DNA-binding domain do they all share?
Glucocorticoid, estrogen, thyroid hormone, progesterone, vitamin D and retinoic acid receptors all share a common DNA-binding domain that forms two zinc fingers.
What happens when there is HIGH glucose and HIGH lactose?
Glucose high = no cAMP-CRP binding: RNA pol is not activated Lactose high = repressor not bound to operon Lac operon is turned OFF due to CRP being unable to activate RNA polymerase, even though the repressor isn't active.
What happens when there is HIGH glucose and LOW lactose?
Glucose high = no cAMP-CRP binding: RNA pol is not activated Lactose low = repressor is bound to operon Lac operon is OFF due to CRP being unable to activate RNA polymerase AND due to the repressor being active
What happens when there is LOW glucose and HIGH lactose
Glucose low = cAMP-CRP binding occurs: RNA pol activated Lactose high = repressor not bound to operon Lac operon is ON due to activation of RNA pol from cAMP-CRP binding AND due to inactivation of repressor
What happens when there is LOW glucose and LOW lactose
Glucose low = cAMP-CRP binding occurs; RNA pol activated Lactose low = repressor is bound to operon Lac operon is OFF due to the repressor being active, even though RNA pol is activated through cAMP-CRP binding
Describe how hypermethylation can cause cancer Describe how hypomethylation can cause cancer
Hypermethylation: In normal cells, tumor suppressor genes lie near hypomethylated CpG islands. If this area gets hypermethylated, transcription of the tumor suppressor gene is repressed, and normal cells will likely become tumor cells. Hypomethylation: In tumor cells, repeat-rich heterochromatin tends to be hypermethylated to promote cell division (these genes are usually turned off). If this area gets hypomethylated, mitotic recombination increases, which decreases the stability of the genome. This genomic instability is a hallmark of tumor cells.
Describe how the glucocorticoid receptor works as a transcription factor
In the cytoplasm, the glucocorticoid receptor is bound to the chaperon protein hsp90, and this complex is inactive. Once a steroid hormone binds, hsp90 dissociates and the activated glucocorticoid receptor migrates to the nucleus. Here it binds to a sequence called glucocorticoid response element that initiates transcription.
What can a lack of methylation induce? What was used to test the role of methylation in gene expression? What does high concentrations of this molecule cause? Name the trade name of the medication containing this compound and what disease it treats.
Lack of methylation can induce fibroblast cells to differentiate into myoblasts that fuse to form muscle cells. Methylation inhibitor 5-azacytidine was used to test the role of methylation in gene expression. 5-azacytidine is a cytidine analog that inhibits DNA methylation, producing hypomethylated DNA. This activates certain genes. At high concentrations, 5-azacytidine is heavily incorporated into DNA and RNA, causing cell death. 5-azacytidine is under the trade name of Vidaza. It has been used to treat myelodysplastic syndrome (MDS).
What molecule recognizes the promoter region? What two sites are contained within the promoter region? What is the function of these two sites?
RNA polymerase recognizes the promoter region. The CRP site and the operator are included in the promoter region. The CRP protein binds cAMP, which causes a conformational change that allows CRP to bind tightly to a specific DNA site in the promoters of the genes it controls. The operator binds a protein that is encoded by the repressor gene, creating a repressor protein that binds to operator site.
Reciprocal crossing over between what two chromosomes produces the Philadelphia chromosome? What is this chromosome responsible for? Describe the BCR promoter and abl genes role in reciprocal crossing over.
Reciprocal crossing over between chromosomes 9 and 22 produces the Philadelphia chromosome, which is responsible for chronic myelogenous leukemia. Chromosome 9's Q site becomes larger, and chromosome 22's Q site becomes smaller after reciprocal crossing over. After translocation, the BCR promoter on chromosome 22 at the abl gene is combined with the abl gene downstream. The abl gene is now under control of BCR promoter The BCR promoter is strong, promoting a higher rate of transcription.
Messing up DNA methylation may cause cancer due to
Reduced tumor suppressor activity Increased mitotic activity Increased genomic instability
Describe the regulatory mechanisms of steroid receptor transcription factors (TFs) and G-protein coupled receptors (GPCR)
Steroid receptors function as transcription factors. When activated by steroid hormones, they bind DNA enhancers and regulate the expression of downstream genes. GPCR functions through the production of second messengers.
If an operon encodes enzymes x, y, and z, which convert substrate A to product B... A is an inducer and B is a co-repressor OR B is an inducer and A is a co-repressor
Substrate A is an inducer and product B is a co-repressor
Fill in the blanks about constitutive vs. regulated gene expression: The _____ of gene expression refers to the abundance of protein or RNA products under given conditions. A gene is ________ expressed when the products are highly abundant, and ______ expressed when the products are low. ___________ vs. _________ gene expression refers to the pattern of expression of the gene. A gene that is __________ expressed does not have significant change in the level of the encoded protein over the period of time for consideration. A gene that is not constitutively expressed can be __-_________ or ____-_________.
The "level" of gene expression refers to the abundance of protein or RNA products under given conditions. A gene is "strongly" expressed when the products are highly abundant, and "weakly" expressed when the products are low. "Constitutive" vs. "regulated" gene expression refers to the pattern of expression of the gene. A gene that is "constitutively" expressed does not have significant change in the level of the encoded protein over the period of time for consideration. *need them all the time A gene that is not constitutively expressed can be "up-regulated" or "down-regulated."
What can be expected to the expression level of a gene downstream of a CpG island?
The expression level of a gene downstream of a CpG island can be down-regulated by methylation.
Describe how the transcription of the lac operon is repressed, and how it is induced
Transcription repressed: (no lactose around) repressor gene upstream of promoter is able to be transcribed into mRNA and translated into a repressor protein. This repressor protein binds to the operator of the lac operon and blocks RNA polymerase from transcribing the operon (cis-acting factor). Transcription induced: (lactose available) lactose binds to the repressor protein, inactivating it. Since the repressor can no longer bind to the operator, beta-galactosidase is free to be synthesized so that lactose can be broken (trans-acting factor).
Describe some regulatory protein types that work together in controlling RNA polymerase at the promoter.
Weak activating protein assembly Strong inhibiting protein Strong activating assembly Silent assembly of regulatory proteins
Describe how the transcription of the trp operon is repressed, and how it is induced
When tryptophan binds to the repressor on the trp operon, it activates the synthesis of another repressor protein. This repressor protein is now able to bind to the promoter to stop synthesis of tryptophan since there's already enough around. When there is no tryptophan to bind to the repressor, additional repressor proteins are not made and synthesis of tryptophan can occur. *Tryptophan is a co-repressor
Besides a DNA binding domain, a transcription factor must have:
an effector domain
Choose one: mRNA decay: only works in regulated gene expression only works in constitutive gene expression may play a role in both constitutive and regulated gene expression
mRNA may play a role in both constitutive and regulated gene expression